Mod Spec® is a mass spectrometry method that is used to measure the relative abundance of > 60 different histone modifications. Mod Spec® is a great starting point to determine if epigenetic changes are occurring in response to disease or treatment. The large number of modifications monitored allows researchers to not only observe expected changes, but to discover unexpected changes, which can be even more informative.
Analyze histone modification changes in:
- Response to epigenetic inhibitors
- Normal vs diseased cells
- Knock-out cells or animals
- Inhibitor treated xenografts
- Human biopsies
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Submit cell pellets or tissue and Active Motif will perform histone extractions, sample preparation, and mass spectrometry using the Thermo Scientific™ TSQ Quantum Ultra™ Triple-Stage Quadrupole Mass Spectrometer. All samples are run in triplicate and data are presented in an easy to understand format. Data delivery typically occurs within 4 - 6 weeks of sample submission.
- Cells required: 2 - 5 million
- Tissue requirements: 25 - 100 mg
|Histone modifications detected by Mod Spec®|
|H1.4: K25UN||H1.4: K25AC||H1.4: K25ME1|
|H1.4: K25ME2||H1.4: K25ME3||^H2A: K5UN|
|^H2A: K5AC||^H2A: K9UN||^H2A: K9AC|
|^H2A: K36UN||^H2A: K36AC||^H2A1: K13UN|
|^H2A1: K13AC||^H2A1: K15UN||^H2A1: K15AC|
|^H2A1: K15UB||^H2A3: K13UN||^H2A3: K13AC|
|^H2A3: K15UN||^H2A3: K15AC||^H2A3: K15UB|
|*H3R2UN: K4UN||*H3R2UN: K4AC||*H3R2UN: K4ME1|
|*H3R2UN: K4ME2||*H3R2UN: K4ME3||H3R2UN: Q5UN|
|H3R2UN: Q5ME1||H3: K9UN||H3: K9AC|
|H3: K9ME1||H3: K9ME2||H3: K9ME3|
|H3: K14UN||H3: K14AC||H3: K18UN|
|H3: K18AC||H3: K18ME1||H3: Q19UN|
|H3: Q19ME1||H3: K23UN||H3: K23AC|
|H3: K23ME1||H3: R42UN||H3: R42ME2|
|H3: R49UN||H3: R49ME2||H3: Q55UN|
|H3: Q55ME1||H3: K56UN||H3: K56AC|
|H3: K56ME1||H3: K64UN||H3: K64AC|
|H3: K79UN||H3: K79AC||H3: K79ME1|
|H3: K79ME2||H3: K79ME3||H3: K122UN|
|H3: K122AC||♦H3.1: K27UN||♦H3.1: K27AC|
|♦H3.1: K27ME1||♦H3.1: K27ME2||♦H3.1: K27ME3|
|♦H3.1: K36UN||♦H3.1: K36AC||♦H3.1: K36ME1|
|♦H3.1: K36ME2||♦H3.1: K36ME3||H3.3: K27UN|
|H3.3: K27AC||H3.3: K27M||H3.3: K27ME1|
|H3.3: K27ME2||H3.3: K27ME3||H3.3: K36UN|
|H3.3: K36AC||H3.3: K36ME1||H3.3: K36ME2|
|H3.3: K36ME3||H4: K5UN||H4: K5AC|
|H4: K8UN||H4: K8AC||H4: K12UN|
|H4: K12AC||H4: K16UN||H4: K16AC|
|H4: K20UN||H4: K20AC||H4: K20ME1|
|H4: K20ME2||H4: K20ME3|
^ Multiple H2A isoforms may contribute to the signal for modifications on H2A.
* H3R2me2 and H3K4me2/3 are mutually exclusive modifications. H3R2 methylation prevents H3K4 methylation. Therefore, H3K4 modifications are reported only on the H3R2 unmodified peptide. For more information, see Nature. 2007 Oct 18; 449(7164):933-7.
♦ H3.2 may contribute to signals for modifications labeled H3.1.
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- Mod Spec® Service Data
- How does Mod Spec® work?
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|Active Motif Epigenetic Services Brochure|
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|Mod Spec Cell Prep Protocol|
Figure 1: Mod Spec® data from control HeLa cells and HeLa cells treated for 7 days with 0.5uM GSK-126.
Figure 2: Mod Spec® data from control HEK293 cells and HEK293 cells treated for 6 hours with 5 mM sodium butyrate (NaB).
How does Mod Spec® work?
- Histones are extracted from samples.
- Derivatization of histones using propionic anhydride.
- Mass spectrometry of proteins requires digestion of the proteins into small peptides before being injected into the mass spectrometer. However, if peptides are too small they can not be separated in the LC step and if too big they are too complex to analyze. The most common method of digestion is with trypsin, an enzyme that cleaves proteins at lysine and arginine residues. This presents a problem when analyzing histones since histone tails are rich in lysine, resulting in digested peptides that are too small. Propionylation, using propionic anhydride, blocks cleavage at lysines while allowing digestion at arginines to occur. The resulting peptides are of the appropriate size for mass spec analysis.
- Histones are digested with trypsin.
- Samples are analyzed on a Triple-Stage Quadrupole Mass Spectrometer.
- Peptides of the appropriate molecular weight are selected.
- Peptides are further fragmented in the collision chamber.
- Peptides fragments of interest are selected and analyzed by mass spectrometry.
- Fragments are analyzed and graphed to show changes between samples.